The interaction of the glycosylphosphatidylinositol (GPI)-anchored cell-surface urokinase receptor (uPAR) and its serine protease ligand urokinase-type plasminogen activator (uPA) is believed to be implicated in nearly every step of tumor formation and progression, including tumorigenesis (1. Shapiro et al. (1997) Urokinase-type plasminogen activator-deficient mice are predisposed to staphylococcal botryomycosis, pleuritis, and effacement of lymphoid follicles. The American journal of pathology 150(1):359-369; the foregoing publication, and each additional publication cited herein, is incorporated herein by reference in its entirety), cell proliferation (2. Gandhari et al. (2006) Urokinase-type plasminogen activator induces proliferation in breast cancer cells. International Journal of Oncology 28(6):1463-1470; 3. Liang et al. (2008) RNAi-mediated downregulation of urokinase plasminogen activator receptor inhibits proliferation, adhesion, migration and invasion in oral cancer cells. Oral Oncol.; 4. Kondraganti et al. (2006) RNAi-mediated downregulation of urokinase plasminogen activator and its receptor in human meningioma cells inhibits tumor invasion and growth. Int J Oncol 28(6):1353-1360), cell migration (5. Prager et al. (2004) Vascular endothelial growth factor receptor-2-induced initial endothelial cell migration depends on the presence of the urokinase receptor. Circ Res 94(12):1562-1570; 6. Schiller et al. (2009) Mannose 6-phosphate/insulin-like growth factor 2 receptor limits cell invasion by controlling alphaVbeta3 integrin expression and proteolytic processing of urokinase-type plasminogen activator receptor. Molecular biology of the cell 20(3):745-756), adhesion (3; 7. Andreasen et al. (1997) The urokinase-type plasminogen activator system in cancer metastasis: A review. Int J Cancer 72(1):1-22), angiogenesis (8. Mignatti & Rifkin (1996) Plasminogen activators and matrix metalloproteinases in angiogenesis. Enzyme & protein 49(1-3):117-137; 9. Rabbani & Mazar (2001) The role of the plasminogen activation system in angiogenesis and metastasis. Surgical oncology clinics of North America 10(2):393-415, x), and invasion (3, 4, 10. Subramanian et al. (2006) siRNA-mediated simultaneous downregulation of uPA and its receptor inhibits angiogenesis and invasiveness triggering apoptosis in breast cancer cells. International Journal of Oncology 28(4):831-839; 11. Kunigal et al. (2007) RNAi-mediated downregulation of urokinase plasminogen activator receptor and matrix metalloprotease-9 in human breast cancer cells results in decreased tumor invasion, angiogenesis and growth. International journal of cancer 121(10):2307-2316). The uPAR•uPA complex formation has been reported to enhance pericellular proteolysis through activation of plasminogen (7), culminating in the active degradation of extracellular matrix (ECM) components. Whereas uPAR is believed to have no inherent signaling capability, it has been reported that the uPAR•uPA complex promotes signaling by actively associating to cell surface receptors such as integrins (12. Wei et al. (1996) Regulation of integrin function by the urokinase receptor. Science 273(5281):1551-1555), receptor tyrosine kinases (RTKs) (13. Kiyan et al. (2005) Urokinase-induced signaling in human vascular smooth muscle cells is mediated by PDGFR-beta. Embo J 24(10):1787-1797; 14. Liu et al. (2002) EGFR is a transducer of the urokinase receptor initiated signal that is required for in vivo growth of a human carcinoma. Cancer cell 1(5):445-457), and G-protein coupled receptors (GPCRs) (15. Resnati et al. (2002) The fibrinolytic receptor for urokinase activates the G protein-coupled chemotactic receptor FPRL1/LXA4R. P Natl Acad Sci USA 99(3):1359-1364). Furthermore, it is believed that uPAR binding to vitronectin components may indicate interactions that extend beyond the cell surface to engage the microenvironment and further promote metastasis.
Compounds capable of disrupting such uPAR-uPA complexes have not been reported (16. Chen et al. (2009) Challenges for Drug Discovery—A Case Study of Urokinase Receptor Inhibition. Combinatorial chemistry & high throughput screening 12(10):961-967). Instead, efforts concentrating on inhibiting the enzymatic activity of uPA have been reported, which may neglect the signaling capabilities of the receptor. It has been reported that the uPAR•uPA complex poses the same challenges that have plagued efforts to inhibit protein-protein interactions with small molecules in the past (17. Arkin & Wells (2004) Small-molecule inhibitors of protein-protein interactions: progressing towards the dream. Nature reviews 3(4):301-317), which include a tight interaction (18. Ploug & Ellis (1994) Structure-function relationships in the receptor for urokinase-type plasminogen activator. Comparison to other members of the Ly-6 family and snake venom alpha-neurotoxins. Febs Lett 349(2):163-168; 19. Gardsvoll et al. (2006) Characterization of the functional epitope on the urokinase receptor. Complete alanine scanning mutagenesis supplemented by chemical cross-linking. The Journal of biological chemistry 281(28):19260-19272), a large interaction surface estimated at 1,318 Å2 (20. Huai et al. (2006) Structure of human urokinase plasminogen activator in complex with its receptor. Science 311(5761):656-659), and inherent flexibility of the target (21. Wells & McClendon (2007) Reaching for high-hanging fruit in drug discovery at protein-protein interfaces. Nature 450(7172):1001-1009).
There exists a need for compounds that are capable of disrupting such uPAR-uPA complexes in the treatment of disease. Without being bound by theory, it is believed herein that small molecules that disrupt hot-spot interactions may be particularly useful for inhibiting protein-protein interactions, such as are present in uPAR-uPA complexes (22. Cerchietti et al. (2010) A Small-Molecule Inhibitor of BCL6 Kills DLBCL Cells In Vitro and In Vivo. Cancer cell 17(4):400-411; 23. Vassilev et al. (2004) In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 303(5659):844-848; 24. Christ et al. (2010) Rational design of small-molecule inhibitors of the LEDGF/p75-integrase interaction and HIV replication. Nature Chemical Biology 6(6):442-448; 25. Oltersdorf et al. (2005) An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435(7042):677-681; 26. Roehrl et al. (2004) Selective inhibition of calcineurin-NFAT signaling by blocking protein-protein interaction with small organic molecules. Proceedings of the National Academy of Sciences of the United States of America 101(20):7554-7559).
Described herein is a computational approach for screening and selecting molecules, including small molecules, that inhibit the uPAR•uPA interaction. Without being bound by theory, it is believed herein that targeting unproductive alternative conformations of uPAR concomitant with disrupting hot-spot interactions at the complex interface (19) may lead to more effective inhibitors of the high-affinity interaction. It is understood herein that it may be advantageous to distinguish those molecules that merely bind to a target, which may be initially identified, from those molecules that are capable of displacing the full protein-protein interaction. Also described herein is the targeting of representative conformers collected from MD simulations by virtual screening of large libraries of compounds. The process includes selecting compounds from the library screen and initially assessing each for activity in a fluorescence polarization assay then in a microtiter-based ELISA. Also described herein is immunofluorescence imaging in tumor cells, which establishes that disruption of uPAR•uPA is also observed in tumor cells. The effect of inhibitors in blocking processes critical in tumor invasion, migration and adhesion of tumor cells, including highly aggressive breast MDA-MB-231 and lung H1299, in cell culture is also described herein.
In one embodiment, described herein are compounds that inhibit interactions of the urokinase receptor, a protein whose interactions are widely believed to promote tumor invasion and metastasis.
In another embodiment, compounds described herein bind at the uPAR/uPA interface, thereby inhibiting the binding of uPA to uPAR and blocking activation of uPA at the cell surface. In another embodiment, compounds described herein that inhibit the uPAR-uPA interaction in vitro. In another embodiment, compounds described herein inhibit the uPAR-uPA interaction in highly aggressive breast and lung tumor cells. In another embodiment, compounds described herein show inhibition of lung and breast cancer invasion, which is a process that drives cancer metastasis in patients. In another embodiment, compounds described herein bind to uPAR on invading cells and block binding of uPA, thus preventing invading cells from invading normal cells, and also preventing cancer cells from migrating.
It has been discovered herein that the compounds described herein inhibit the uPAR-uPA protein-protein interaction. In one embodiment, compounds described herein are useful as chemical probes for this interaction. In another embodiment, compounds described herein are useful as leads for the development of therapeutic agents to block metastasis in the clinic. In another embodiment, compounds described herein are useful in the treatment of cancer.
In one illustrative embodiment of the invention, compounds of the following formula are described herein:
or pharmaceutically acceptable salts thereof, wherein RA, X, and Y are as defined below in the various illustrative embodiments herein.
In addition, various genera and subgenera of each of RA, X, and Y are described herein. It is to be understood that all possible combinations of the various genera and subgenera of each of RA, X, and Y described herein represent additional illustrative embodiments of compounds of the invention described herein. It is to be further understood that each of those additional illustrative embodiments of compounds may be used in any of the compositions, methods, and/or uses described herein.
In another embodiment, pharmaceutical compositions containing one or more of the compounds are also described herein. In one aspect, the compositions include a therapeutically effective amount of the one or more compounds for treating a patient with cancer. It is to be understood that the compositions may include other components and/or ingredients, including, but not limited to, other therapeutically active compounds, and/or one or more carriers, diluents, excipients, and the like, and all combinations of the foregoing.
In another embodiment, methods for using the compounds and pharmaceutical compositions for treating patients with cancer are also described herein. In one aspect, the methods include the step of administering one or more of the compounds and/or compositions described herein to a patient with cancer. In another aspect, the methods include administering a therapeutically effective amount of the one or more compounds and/or compositions described herein for treating patients with cancer.
In another embodiment, uses of the compounds and compositions in the manufacture of a medicament for treating patients with cancer are also described herein. In one aspect, the medicaments include a therapeutically effective amount of the one or more compounds and/or compositions for treating a patient with cancer.
It is appreciated herein that the compounds described herein may be used alone or in combination with other compounds useful for treating cancer, including those compounds that may be therapeutically effective by the same or different modes of action. In addition, it is appreciated herein that the compounds described herein may be used in combination with other compounds that are administered to treat other symptoms of cancer.